Expression of, and Preliminary Biophysical Characterization of a Minimal Binding Domain Peptide of TSC2 That Binds to the Small GTPase Rheb

by Liu, Huimin; Adams, Paul

Rheb (Ras homolog enriched in brain) is a recently identified Ras protein cycles between GTP-and GDP-bound states similarly to other Ras proteins. An important interaction between Rheb and the Tuberous Sclerosis complex proteins (TSC1/2) mediates the mammalian Target Of Rapamycin (mTOR) pathway, which regulates cell growth, energy and nutrient levels. TSC2 contains a GTPase-binding domain near its C-terminus that contains a highly conserved 15-amino acid region. Mutations in this region has resulted in altered GTPase activity towards Rheb, suggesting that the 15 amino acid conserved region is vital for this interaction. To date, mol. details of this important Ras-effector interaction are unknown. We present here a preliminary characterization of an interaction of Rheb with a minimal GTPase-binding domain peptide deriv. of TSC2. A series of peptides truncated from the C-terminal region of TSC2 encompassing the GTPase-binding domain were expressed in bacterial cell lines. A peptide of 37 amino acids (TSC2-37) has demonstrated similar binding affinity to Rheb in-vitro to a 900 amino acid C-terminal deriv. of TSC2. Isothermal titrn. calorimetry was used to examine the binding affinity of a synthetic peptide consisting of only the15 amino acids of the conserved GTPase-binding region of TSC2 for Rheb to highlight the importance of the conserved region in binding. These results represent a first step towards the biophys. characterization of the Rheb-TSC2 complex that is expected to provide mol. details that can be translated to its biol. function.